Purification of Delicate
Compounds with RediSep Rf
Gold® Diol and Cyano Columns
Chromatography Application Note
AN100
Abstract
Many compounds are difficult to purify because they
decompose on the column. An epoxide compound was
synthesized and purified with a RediSep Rf Gold Diol and
RediSep Rf Gold Cyano column. The epoxide makes an
excellent model for other unstable compounds as epoxides commonly decompose on silica gel. Unstable
compounds are often purified using a series of
liquid-liquid extractions. Purifying such compounds on
a column where the amount of solvent used and evaporation is reduced is desirable.
The RediSep Rf Gold Diol and RediSep Rf Gold Cyano
columns are useful for purifying delicate compounds
because they are less active than silica gel. They are
both run as normal phase columns; normal phase solvents usually have a lower boiling point than reverse
phase solvents. Using lower boiling solvents decreases
the heat necessary to evaporate the solvents and
reduces evaporation time after purification, thus
reducing the time fragile compounds are exposed to
heat.
Experimental and Results
SynthesisofTrans‐anisoleoxide
Trans-anisole oxide was prepared according to a published procedure1. Trans- anethole
(p-Methoxy-trans--methylstyrene, 0.50 g, 3.4 mmol) was
dissolved in 10 ml dichloromethane. This solution was
then added to 10% aqueous Na2CO3 (20 ml) in an ice
bath. A solution of m-chloroperoxybenzoic acid (1.4 g,
5.7 mmol dissolved in 20 ml dichloromethane) was
added next at a rate 0.5 ml/min with vigorous stirring to
the trans-anethole mixture. After the addition, the mixture was allowed to stir for an additional 20 minutes.
The organic layer was separated and washed with
10% Na2CO3 (5x25 ml). The organic layer was dried over
Na2SO4 and evaporated to dryness (45 °C bath temperature) to obtain a clear oil (0.5567 g). This mixture was
used for the purification on a RediSep Rf Gold Diol
column. The reaction used for the RediSep Rf Gold
Cyano column yielded 0.5712 g of oil.
1. Centko, R.S.; Mohan, R.S. The Discovery-Oriented Approach to
Organic Chemistry. 4. Epoxidation of p-Methoxy-trans--methylstyrene. J. Chem. Ed. 2001, 74(1), 77-79.
Figure 1: Synthesis of trans-anesole oxide
Purification on RediSep Rf Gold Diol
A reusable diol solid load cartridge was made by
filling a 5 g “empty” solid load cartridge (PN 69-3873-235)
with 2.5 g RediSep bulk diol media (PN 60-5394-502). The
reaction mixture was dissolved in dichloromethane and
placed in the diol solid load cartridge. The use of diol
media in the solid load cartridge provided a compatible
media for elution for the chromatography. The dichloromethane was evaporated by placing the solid load
cartridge on the CombiFlash® Rf+ (PN 68-5230-021)
instrument and blowing air through the cartridge with
the air-purge feature via manual control. As illustrated in
Chromatography Application Notes 882 and 893, loading
the sample on a solid load cartridge improves resolution. The diol packing ensures a compatible adsorption
surface and minimized compound decomposition.
The mixture was eluted with a hexane-ethyl acetate
gradient (Figure 2) using a 50 g RediSep Rf Gold Diol
column (PN 69-2203-517). Fractions were collected at
280 nm and evaporated into tared flasks at 45 °C.
2. http://www.isco.com/WebProductFiles/Applications/101/
Application_Notes/AN88_C18_Flash_Column_Loading.pdf retrieved
21 June 2014.
3. http://www.isco.com/WebProductFiles/Applications/101/
Application_Notes/
AN29_Overview_of_Silica_Column_Sample_Loading_Techniques.p
df retrieved 21 June 2014.
Chromatography Application Note AN100
2.0
100
90
80
70
60
1.0
50
40
30
0.5
Ethyl Acetate (%)
Absorbance (280 nm)
1.5
20
10
0.0
0
2
4
6
8
10
12
14
16
0
Time (CV)
Figure 2: Purification of trans-anisole oxide with
RediSep Rf Gold Diol column
Table 1: Compound Recovery
Fraction
Recovered Mass (g)
1
2
3
4
5
6
Total:
0.0549
0.2766
0.1030
0.0139
0.0136
0.0543
0.5163
The desired reaction product eluted in fractions 2
and 3. The compound recovery in these fractions was
0.3796 g, or 68% reaction yield, compound identity was
verified by NMR and comparison with the spectra
reported in Reference 1.
Purification on RediSep Rf Gold Cyano
A reusable cyano solid load cartridge was made by
filling a 5 g “empty” solid load cartridge (PN 69-3873-235)
with 2.5 g RediSep bulk cyano media (PN 60-5394-501).
The reaction mixture was dissolved in dichloromethane
and placed in the cyano solid load cartridge. The dichloromethane was evaporated by placing the solid load
cartridge on the CombiFlash® Rf+ (PN 68-5230-021)
system and blowing air through the cartridge with the
air-purge feature via manual control. The cyano solid
load cartridge ensures a compatible adsorption surface
and minimizes compound decomposition. The mixture
was eluted with a hexane-ethyl acetate gradient (Figure
3) using a 50 g RediSep Rf Gold Cyano column (PN
69-2203-497). Fractions were collected at 280 nm and
evaporated into tared flasks at 45 °C.
Chromatography Application Note AN100
2.5
100
90
80
70
1.5
60
50
1.0
40
30
0.5
Ethyl Acetate (%)
Absorbance (280 nm)
2.0
20
10
0.0
0
2
4
6
8
10
12
14
16
0
Time (CV)
Figure 3: Purification of trans-anisole oxide with a
RediSep Rf Gold Cyano column
Table 2: Compound Recovery
Fraction
Recovered Mass (g)
1
2
3
4
5
6
7
Total:
0.0575
0.3012
0.421
0.0107
0.0076
0.0510
0.0187
0.4888
form an ester that irreversibly binds to the silica. No
material was found to elute from silica. The cyano and
diol groups are bonded to the silica media which protects the compound by preventing reaction of the
epoxide with acidic silanol groups while providing a surface with the required selectivity to purify the mixture
running as normal phase columns. The use of
low-boiling normal phase solvents allows rapid evaporation of fractions at a moderate temperate compared to
reverse phase solvents, further reducing the stress on
fragile compounds.
The desired reaction product eluted in fraction 2.
The compound recovery in this fraction was 0.3012 g, or
55% reaction yield, verified by NMR; the PMR spectrum
matched that from Reference 1.
Conclusion
RediSep Rf Gold Diol and RediSep Rf Gold Cyano columns provide viable alternative methods for the
purification of delicate compounds, such as epoxides,
that often decompose on silica gel. Acidic silanols on
the chromatographic media likely open the epoxide to
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